The mitochondrial theory of senescence suggests that lifelong damage to mitochondrial DNA (mtDNA) underlies the cellular senescence of aging and contributes to the pathogenesis of some late-onset diseases like Alzheimer's. This theory rests on a wealth of data showing that the frequency of mtDNA mutations rises with age while mitochondrial respiration declines. It is also supported by a plausible mechanism for the generation of those mutations: namely, that toxic oxygen metabolites, the natural by-products of mitochondrial respiration, react with and damage mtDNA. There is little direct evidence, however, that high levels of mtDNA mutations are causally related to either the aging process itself or age-related diseases like Alzheimer's. Since nearly all the data is correlative, an alternate interpretation is that the mutations are primarily a consequence of aging. We propose to construct an animal model in which to test the hypothesis that a rising frequency of mtDNA mutations is an important factor in the aging process. Transgenic mice will be made in which mtDNA mutations accumulate with age at an accelerated rate. These mice will contain a transgene expressing. a mutant form of the mtDNA polymerase which lacks proofreading. Since the mammalian mtDNA polymerase appears to have no role in nuclear DNA replication, mice expressing the mutant transgene are predicted to show decreased fidelity of only mtDNA replication. The decreased fidelity will lead, in turn, to an accelerated accumulation of mtDNA mutations in the transgenic mice. The goal of this pilot project proposal is to construct such transgenic mice. We do not intend to knock out endogenous DNA polymerase activity in these mice; rather, we will rely on a sufficient level of expression of the mutant transgene to establish biochemical dominance by mass action. Preliminary investigations in cell lines will be performed to determine the appropriate expression level of the mutant DNA polymerase. Therefore, the Specific Aims in this proposal are: 1. - To construct cell lines expressing a proof-reading deficient DNA polymerase 2. - To construct transgenic mice having a high frequency of mtDNA mutations